The present invention relates to an oligosaccharide derivative and a process for producing the same. More specifically, the invention relates to an oligofucose or oligorhamnose derivative, which is effective for prevention and treatment of inflammation and ulceration on the mucosa of gastric organs such as stomach and duodenum.
Conventional antiulcer agents include those for controlling gastric juice secretion, such as H2-blockers and proton pump inhibitors. For the purpose of active therapeutic treatment of gastritis and gastric mucosal cells, use of prostaglandins or basic fibroblast growth factors have recently been studied. These pharmaceutical agents, on the one hand, exert efficacious therapeutic effects. On the other hand, however, these pharmaceutical agents potentially cause the onset of ulcer, specifically the recurrence of ulcer, for which a specific bacterial species Helicobacter pylori is responsible. Therefore, it is remarked that gastric ulcers should essentially be treated, including the disinfection of the bacterial species (Masahiro Asaka, xe2x80x9cHelicobacter pylon and disorders of gastric mucosal membranexe2x80x9d, Sentan-Igaku-Sha (Top Medicine Press), Jul. 1, 1995).
It has aaditionally been known that microbial cells of certain species of Bifidobacteria or lactic acid bacteria and polysaccharides or oligosaccharides prepared from these microbial cells, Phaeophyceae Chordariales nemacystus or green laver (Monostroma nitidum) are effective in not only the prevention of ulceration but also in the promotion of the healing thereof, and thus are useful as antiulcer agents (JP-A-6-247861; JP-A-7-138166) . These polysaccharides and oligosaccharides primarily comprise fucose and/or rhamnose. Specifically, fucoidan, a fucose polymer, exerts effects to prevent ulcer formation and to promote the healing of urceration and additionally exerts an action to inhibit the adhesion of H. pylori. Nevertheless, these polysaccharides and oligosaccharides per se are not satisfactory in terms of their antiulcer activity. Furthermore, it is difficult to recover these polysaccharides and oligosaccharides at homogenous states including the homogeneity of their molecular weights.
It is a primary object of the invention to provide an oligosaccharide derivative having higher homogeneity and greater antiulcer effects than those of the aforementioned conventional polysaccharides, and another object of the present invention is to provide a process for producing the same.
According to an embodiment of the present invention, there is provided an oligosaccharide derivative represented by the following general formula:
Y1xe2x80x94OCH(CH2NHR)2
wherein, Y1 represents an oligofucose at a polymerization number of 2 to 20, where the hydroxyl groups may or may not be partially modified in the form of sulfate ester, and R represents phenyl group, a higher alkylphenyl group, a higher alkyl group or xe2x80x94(CH2)nxe2x80x94NHX wherein n is an integer of 1 to 10 and X represents a higher alkanoyl group or an alkylamino group with or without substituents.
According to another embodiment of the present invention, there is also provided an oligosaccharide derivative represented by the following general formula:
Y2xe2x80x94OCH (CH2NHR)2
wherein, Y2 represents an oligorhamnose at a polymerization number of 2 to 20, where the hydroxyl groups may or may not be partially modified in the form of sulfate ester, and R represents phenyl group, a higher alkylphenyl group, a higher alkyl group or xe2x80x94(CH2)nxe2x80x94NHX wherein n is an integer of 1 to 10 and X represents a higher alkanoyl group or an alkylamino group with or without substituents.
These oligosaccharide derivatives have various properties including excellent antiulcer effect.
The present invention also provides a process for producing an oligosaccharide derivative comprising the steps of converting the sugar residue at the reducing end of the oligofucose or oligorhamnose to aldehyde group through oxidative degradation, generating a Schiff base by allowing the aldehyde group to react with at least one of corresponding alkylamines and allylamines, and reducing the Schiff base to obtain an oligosaccharide derivative.
That is, in the process according to the present invention, fucoidan (fucose polysaccharide) or rhamnan (rhamnose polysaccharide) is modified into an oligosaccharide (an oligomer comprising about 3 to 20 molecules of fucose or rhamnose) through acid treatment, and then, the oligosaccharide is subjected to oxidation with periodate, reaction with corresponding amines, and reductive treatment
According to another aspect of the invention, there is provided an inhibitor for H. pylori which comprises the oligosaccharide derivative, namely oligofucose derivative and/or oligorhamnose derivative according to the present invention.
According to still another aspect of the present invention, there is provided an agent for preventing and therapeutically treatment of gastric ulcer, said agent comprising the oligosaccharide derivative, namely oligofucose derivative and/or oligorhamnose derivative according to the present invention.
According to one embodiment of the present invention, the process for producing an oligosaccharide derivative comprises the following steps 1 to 8.
Step 1: Using known extraction methods, polysaccharides are extracted from marine algae (such as Phaeophyceae Chordariales nemacystus, Hydrilla, Fucus, and Monostroma nitidum) containing fucoidan, rhamnan or rhamnan sulfate.
Step 2: The recovered polysaccharides are dissolved in a solution of hydrochloric acid or trifluoroacetate of about 0.075 N to 0.1 N; the mixture is heated at 100xc2x0 C. for 10 to 20 minutes, to modify the polysaccharides into the form of oligosaccharides. After the treatment, the resulting reaction mixture is neutralized with sodium hydroxide, followed by addition of NaBH4 to the neutraized mixture for reductive treatment at ambient temperature or 4xc2x0 C. for 16 hours.
Step 3: The solution of the oligosaccharides in the alditol form as recovered by the procedures of the step 2 is desalted by dialysis (a fractionating molecular weight of 500) or electro-dialysis or by using an ion exchange resin.
Step 4: To the solution recovered at the step 3, sodium metaperiodate is added for reaction in a vessel immersed in an ice bath for about one hour. Herein, an oligosaccharide in a structure such that sugars except the sugars at the reducing ends or the non-reducing ends are never oxidized, for example (1xe2x86x923) oligosaccharide, may satisfactorily be subjected to the reaction for a longer period of time. Ethylene glycol at a volume excessive to periodic acid is added to the reaction solution, for additional reaction for about one hour. The resulting solution is subjected to desalting in the same manner as in the step 3. By the procedure, an oligosaccharide derivative (in liquid) with an aldehyde group at the end can be recovered.
Step 5: Acetic acid is added to the solution from the step 4 to a final concentration of 0.5 M, which is subjected to reaction at ambient temperature for 20 hours. The reaction solution is dialyzed against a dialysis membrane of a fractionating molecular weight of 500; the inner dialysis residue solution is freeze-dried to recover an objective oligosaccharide. Additionally, the oligosaccharide fraction can be desalted by dialysis. Alternatively, the oligosaccharide fraction can be fractionated and desalted by chromatography on an active-charcoal column and gel filtration to prepare a fraction of an appropriate molecular weight.
Step 6: The oligosaccharide fraction recovered at the step 5 is dissolved in an aqueous solution containing 40% to 50% propanol, followed by addition of allylamines or alkylamines, for reaction at 45xc2x0 C. for 2 hours, to prepare a Schiff base. In this case, any substance (dimethyl sulfoxide, dimethylformamide, etc.) capable of dissolving oligosaccharides and alkylamines may satisfactorily be used as the solvent. Then, any alkylamine and any allylamine may satisfactorily be selected, with no specific limitation, for use in the reaction; preference is given to anilines with or without substituents and higher alkylamines with or without substituents, wherein the substituents include for example alkyl groups, alkanoylamino groups and alkylamino groups.
Step 7: Volan trimethylamine is added to the solution recovered at the step 6, to reduce the Schiff base at 45xc2x0 C. for 20 hours. Reducing agents satisfying the purpose (for example, Volan dimethylamine, NaCNBH4, NaBH4, etc.), in addition to Volan trimethylamine, can be used appropriately.
Step 8: After the termination of the reduction at the step 7, the solvent is distilled off by means of an evaporator and the like; by subsequently partitioning the resulting solution in a mixture solution of chloroform:methanol:water=2:1:1, the resulting aqueous phase is collected. By rinsing the aqueous phase in chloroform and freeze-drying the aqueous phase, an oligosaccharide derivative comprising an oligosaccharide-alkylamine complex can be recovered. Depending on the properties of the alkylamine, appropriate solvents may be selected and used for the partition and extraction.
It is verified that the oligosaccharide derivative according to the present invention exerts the effect to inhibit the adhesion of H. pylori as a causative bacterial species of gastric ulcer and the effect to therapeutically treat ulcer induced by acetic acid.
According to the present invention, the pharmaceutical agent for preventing and therapeutically treatment of gastric ulcer can be prepared in pharmaceutical dosage forms appropriately selected, which can be administered at an amount appropriately selected. Generally, however, the pharmaceutical agent is blended with a pharmaceutically acceptable carrier in liquid or solid, followed by addition of solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrators, and lubricants and the like, and the resulting mixture is formulated as tablets, granules, powders, powdery formulations, capsules and the like for use. The dose of the pharmaceutical agent appropriately ranges from about 0.1 to 10 mg/kg, preferably from 0.3 to 3 mg/kg, for most adults per day.